Alzheimer's disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimate death. Alzheimer's disease is the leading cause of dementia in the elderly, affecting 5-10% of the population over the age of 65 years (Jorm A, A Guide to the Understanding of Alzheimer's Disease and Related Disorders, University Press, New York, 1987.). In AD, the parts of the brain essential for cognitive processes such as memory, attention, language, and reasoning degenerate. AD is characterized by the deposition and accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein, Aβ (Glenner G G, and C W Wong. Biochem. Biophys. Res. Comm. 120:885-890, 1984., Husby G, et al. Bull WHO 71:105-108, 1993., Masters C L, et al. Proc. Natl. Acad. Sc. USA 82:4245-4249, 1985.). Aβ is derived from larger precursor proteins termed beta-amyloid precursor proteins (or APPs) of which there are several alternatively spliced variants. The most abundant forms of APPs include proteins consisting of 695, 751 and 770 amino acids (Kitaguchi N, et al. Nature 331:530-532, 1988. Ponte P, et al. Nature 331:525-527, 1988., Tanzi R E, et al. Nature 331:528-530, 1988.). The small Aβ peptide is a major component, which makes up the amyloid deposits of “plaques” in the brains of patients with AD either as extracellular amyloid plaques or in blood vessel walls in the parenchyma. In addition, AD is characterized by the presence of numerous neurofibrillary “tangles”, consisting of paired helical filaments (PHFs) that abnormally accumulate in the neuronal cytoplasm (Grundke-Iqbal I, et al. Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986., Kosik K S, et al. Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986., Lee V M Y, et al. Science 251:675-678, 1991.). The pathological hallmarks of AD are therefore the presence of “plaques” and “tangles” with amyloid being deposited in the central core of plaques. The other major type of lesion found in AD brain is the accumulation of amyloid in the walls of blood vessels, both within the brain parenchyma and in the walls of meningeal vessels that lie outside the brain. Aβ amyloid formation, deposition, accumulation and persistence are believed to play a central role in AD pathogenesis by contributing to neuronal loss and memory dysfunction. The primary factor(s) causing amyloid plaque and NFT accumulation leading to the pathogenesis of AD is not known.
Previous studies indicate that the accumulation of Aβ and amyloid is indeed a causative factor for AD. Aβ in cell culture causes degeneration of nerve cells within short periods of time (Pike C J, et al. Br. Res. 563:311-314, 1991., Pike C J, et al. J. Neurochem. 64:253-265, 1995.). Aβ has been found to be neurotoxic in slice cultures of hippocampus (Harrigan M R, et al. Neurobiol. Aging 16:779-789, 1995.) and induces nerve cell death in some forms of transgenic mice (Games D, et al. Nature 373:523-527, 1995., Hsiao K, et al. Science 274:99-102, 1996, Sturchler-Pierrat C, et al. Proc. Natl. Acad. Sci. 94:13287-13292, 1997.). Previous studies utilizing amyloid plaque producing transgenic mice also clearly demonstrate a direct correlation between increased amyloid plaque burden and behavioral deficits in memory tasks (Choi P Y, et al. Neuroscience Meeting, Orlando, Fla., November 2002., Janus C, et al. Nature 408:979-982, 2000., Morgan D, et al. Nature 408:982-985, 2000.). Probably the most convincing evidence that Aβ amyloid is directly involved in the pathogenesis of AD comes from genetic studies in which the production of Aβ resulted from mutations in the APP gene (Haas C, et al. Nature Med. 1:1291-1296, 1995., Murrell J, et al. Science 254:97-99, 1991., Van Broeckhoven C, et al. Science 248:1120-1122, 1990.), and duplication of the APP locus (Rovelet-Lecrux et al., Nature Genetics, 38:24-26, 2006).
Important amyloid co-factors that may play a role in the pathogenesis of AD are specific proteoglycans (PGs) and glycosaminoglycans (GAGs). Previous studies demonstrated that particular heparan sulfate proteoglycans (HSPGs) including perlecan, syndecan-2, glypican, and agrin are specifically immunolocalized to Aβ-containing amyloid plaques and/or cerebrovascular amyloid deposits in AD brain (Perlmutter L S, et al. Br. Res. 508:13-19, 1990., Snow A D, et al. Am. J. Path. 133:456-463, 1988., Snow A D, and T N Wight, Neurobiol. Aging 10:481-497, 1989., Snow A D, et al. Am. J. Path. 137:1253-1270, 1990., Snow A D, et al. Neuron 12: 219-234, 1994., Su J H , et al. Neurosc. 51:801-813, 1992., Van Gool D, et al. Dementia 4:308-314, 1993., Van Horssen J, et al. Lancet 2:482-492, 2003, Castillo G M, et al. J. Neurochem. 69:2452-2465, 1997., Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883, 1991., Snow A D, et al. Am. J. Path. 144:337-347, 1994., Snow A D, et al. Arch. Biochem. Biophys. 320:84-95, 1995, Lashley T, et al. Neuropath. Appl. 32:492-504, 2006., Verbeek M M, et al. Am. J. Path. 155:2115-2125,1999, Lashley T, et al. Neuropath. Appl. 32:492-504, 2006., Verbeek M M, et al. Am. J. Path. 155:2115-2125,1999., Watson D J, et al. J. Biol. Chem. 272:31617-31624, 1997, Cotman S L, et al. Mol Cell. Neurosc. 15:183-198, 2000., Lashley T, et al. Neuropath. Appl. 32:492-504, 2006., Schultz J G, et al. Europ. J. Neuorsc. 10:2085-2093, 1998., Verbeek M M, et al. Am. J. Path. 155:2115-2125,1999., Watanabe N, et al. FASEB J. published online, Apr. 14, 2004., Watson D J, et al. J. Biol. Chem. 272:31617-31624, 1997). These HSPGs also accumulate in transgenic mice overexpressing beta-amyloid precursor protein (APP) and accumulate in brain concurrent with initial Aβ accumulation and deposition (Cummings J A, et al. Annual Meeting of Neuroscience, Washington, DC, Nov 2005, Snow A D, et al. 8th International Conference on Alzheimer's and Parkinson's disease, Salzburg, Austria, March 2007). It is believed that HSPGs facilitate amyloid deposition and/or promote the persistence of amyloid by inhibiting clearance mechanisms (Snow A D, and T N Wight, Neurobiol. Aging 10:481-497, 1989.).Consistent with this hypothesis in vitro studies have revealed that HSPGs such as perlecan (Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883, 1991., Snow A D, et al. J. Histochem. Cytochem. 40:105-113, 1992., Snow A D, et al. Arch. Biochem. Biophys. 320:84-95, 1995.), agrin (Cotman S L, et al. Mol Cell. Neurosc. 15:183-198, 2000.) and glypican (Watson D J, et al. J. Biol. Chem. 272:31617-31624, 1997.) can bind with high affinity to Aβ and APPs (Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883, 1991.). Additionally, in vitro and cell culture studies demonstrate that HSPGs protect Aβ from protease degradation (Gupta-Bansal R, et al. J. Biol. Chem. 270:18666-18671, 1995., Nguyen B P, et al. Annual Meeting of Neuroscience, New Orleans, LO November 2003., Snow A D, et al. Neuron 12: 219-234, 1994.), supporting a role for HSPGs in inhibition of Aβ-degradation and removal in vivo. All of these studies implicate HSPGs as important co-factors postulated to lead to the accumulation and persistence of Aβ. HSPGs are also specifically co-localized to the PHFs in NFTs in AD brain (Snow A D, et al. Acta Neuropath. 78:113-123, 1989., Snow A D and G M Castillo. Amyloid: Int. J. Exp. Clin. Invest. 4:135-141, 1997.). An alternative hypothesis is that PG's may affect APP processing. Our results suggest that syndecan-2 splice variants interfere with β-secretase cleavage of APP which may lead to a reduction in Aβ levels. Studies have also demonstrated that highly sulfated GAGs such as heparan sulfate can induce tau protein to adopt PHF formation identical to that observed in AD brain (Friedrich M V, et al. J. Biol. Chem. 294:259-270, 1999., Goedert M, et al. Nature 383:550-553, 1996., Hasegawa M, et al. J. Biol. Chem. 272:33118-33124, 1997., Perez M, et al. J. Neurochem. 67:1183-1190, 1996.). Our results also support that syndecan-2 splice variants may be relevant to tau NFT formation. Therefore, HSPGs may play an important role in the pathology of AD.
Proteoglycans (PGs) usually consist of a protein core to which are covalently attached one or more glycosaminoglycan (GAG) chains. GAGs consist of a repeating disaccharide unit containing a hexuronic acid (either glucuronic acid or iduronic acid) or hexosamine (glucosamine or galactosamine) (reviewed in Snow A D, and T N Wight. Neurobiol. Aging 10:481-497, 1989). Different classes of GAGs include the highly sulfated heparin and heparan sulfate, and the less sulfated keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and the non-sulfated hyaluronic acid (reviewed in Snow A D, and T N Wight. Neurobiol. Aging 10:481-497, 1989). At least 4 different classes of PGs have been shown to be present in AD brain. These include heparan sulfate proteoglycans (HSPGs) (Perlmutter L S, et al. Br. Res. 508:13-19, 1990., Snow A D, et al. Am. J. Path. 133:456-463, 1988., Snow A D, and T N Wight. Neurobiol. Aging 10:481-497, 1989., Snow A D, et al. Am. J. Path. 137:1253-1270, 1990., Snow A D, et al. Neuron 12: 219-234, 1994., Su J H , et al. Neurosc. 51:801-813, 1992., Van Gool D, et al. Dementia 4:308-314, 1993. Van Horssen J, P et al. Lancet 2:482-492, 2003.), dermatan sulfate PGs (Snow A D, et al. J. Histochem. Cytochem. 40:105-113, 1992), chondroitin sulfate PGs (DeWitt D A, et al. Exp. Neurol. 121:149-152, 1993.) and keratan sulfate PGs (Snow A D, et al. Exp. Neurol. 138:305-317, 1996.). Of all these different PGs, evidence indicated that only the HSPGs are specifically immunolocalized to the Aβ-containing fibrils both in the amyloid plaques and in the cerebrovascular amyloid deposits in AD brain (Perlmutter L S, et al. Br. Res. 508:13-19, 1990, Snow A D, et al. Am. J. Path. 133:456-463, 1988, Snow A D, and T N Wight. Neurobiol. Aging 10:481-497, 1989., Snow A D, et al. Am. J. Path. 137:1253-1270, 1990, Snow A D, et al. Neuron 12: 219-234, 1994., Su J H , et al. Neurosc. 51:801-813, 1992, Van Gool D, et al. Dementia 4:308-314, 1993. Van Horssen J, et al. Lancet 2:482-492, 2003.). Particular HSPGs that have been immunolocalized or identified within Aβ-amyloid deposits in AD brain include perlecan (Castillo et al. J. Neurochem. 69:2452-2465, 1997, Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883, 1991, Snow A D, et al. Am. J. Path. 144:337-347, 1994, Snow A D, et al. Arch. Biochem. Biophys. 320:84-95, 1995), syndecan-2 (Lashley T, et al. Neuropath. Appl. 32:492-504, 2006, Verbeek M M, et al. Am. J. Path. 155:2115-2125, 1999), agrin (Cotman S L, et al. Mol Cell. Neurosc. 15:183-198, 2000, Lashley T, et al. Neuropath. Appl. 32:492-504, 2006, Verbeek M M, et al. Am. J. Path. 155:2115-2125,1999), and glypican (Lashley T, et al. Neuropath. Appl. 32:492-504, 2006, Schultz J G, et al. Europ. J. Neuorsc. 10:2085-2093, 1998, Verbeek M M, et al. Am. J. Path. 155:2115-2125,1999., Watanabe N, et al. FASEB J. published online, Apr. 14, 2004., Watson D J, et al. J. Biol. Chem. 272:31617-31624, 1997.). Our own studies indicate that HSPGs, such as perlecan (which consists of a ˜400 kDa core protein with 3 heparan sulfate GAG chains attached) are integral parts of amyloid deposits in AD brain. Perlecan is present in isolated amyloid plaque core preparations derived from AD brain as determined by positive immunostaining and western blotting with specific perlecan core protein antibodies (Castillo G M, et al. Soc. Neurosc. Abstr. 22:1172, 1996, Castillo G M, et al. 6th International Conference on Alzheimer's Disease and Related Disorders, Amsterdam, July 1998). Perlecan, syndecan-2, glypican and agrin all not only co-localized to Aβ-amyloid deposits in AD brain, but are also present and co-immunolocalized to amyloid plaques in APP transgenic mice (Cummings J A, et al. Annual Meeting of Neuroscience, Washington, DC, November 2005, Snow A D, et al. 8th International Conference on Alzheimer's and Parkinson's disease, Salzburg, Austria, March 2007). In fact, HS GAGs accumulate in APP mouse brain concurrent and co-localized with initial Aβ accumulation and deposition in brain tissue (Cummings J A, et al. Annual Meeting of Neuroscience, Washington, DC, November 2005, Snow A D, et al. 8th International Conference on Alzheimer's and Parkinson's disease, Salzburg, Austria, March 2007). HSPG immunoreactivity is localized to diffuse plaques in AD (Snow A D, et al. Am. J. Path. 133:456-463, 1988, Snow A D, et al. Am. J. Path. 137:1253-1270, 1990., Snow A D, et al. Am. J. Path. 144:337-347, 1994.) and Down's syndrome brain (Snow A D, et al. Am. J. Path. 137:1253-1270, 1990.) suggesting that this particular class of PGs may-in fact represent a primary initiating factor leading to Aβ accumulation and persistence. Consistent with this hypothesis is the observation that in very young Down's syndrome brain (as early as 1 day after birth), marked HS accumulation in neuronal cytoplasm occurs prior and much earlier than the first appearance of Aβ-deposition (in neurons and later in the matrix) and fibrillar amyloid (Snow A D, et al. Am. J. Path. 137:1253-1270, 1990.). In other types of amyloidosis (such as systemic AA amyloidosis) where the temporal relationship in the experimental mouse model has been extensively studied, it is clear that an increase in gene expression of specific HSPGs, such as perlecan, occurs prior to AA amyloid formation and deposition in tissues (Ailles L, et al., Lab. Invest. 69:443-448, 1993, Elimova E, et al. FASEB J. 18:1749-1751, 2004 , Snow A D, and R Kisilevsky, Lab. Invest. 53:37-44, 1985). Furthermore, heparanase overexpressing transgenic mice that cause a decrease in HS accumulation renders mice resistant to induction of systemic AA amyloidosis (Li J P, et al. Proc. Natl. Acad. Sc. 102:6473-6477, 2005) further supporting an important role of HSPGs for the induction of amyloidosis.
Perlecan is a large HSPG normally present on all basement membranes, consisting of 94 exons, coding for a large ˜470 kDa protein core. Perlecan core protein contains a cluster of 3 GAG attachment sites in domain I (Dolan M, et al., J. Biol. Chem. 272:4316-4322, 1997, Murdoch A D, et al., J. Biol. Chem. 267:8544-8557, 1992). Possible splice variants of perlecan have been reported for mammalian perlecan (Joseph S J, et al., Develop. 122:3443-3452, 1996.). Syndecan-2 is one of four members of this single-pass transmembrane family in vertebrates (Kramer K L, and H J Yost, Ann. Rev. Gen. 37:461-484, 2003). The ˜22 kDa core protein is organized into 3 regions: the N-terminal ectodomain containing a signal sequence, followed by 3 predicted GAG attachment sites, a transmembrane domain and a highly conserved cytoplasmic domain (reviewed in Essner J J, et al. Int. J. Biochem. Cell Biol. 38:152-156, 2006).
The HSPGs, perlecan (Castillo G M, et al. J. Neurochem. 69:2452-2465, 1997., Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883, 1991, Snow A D, et al. Am. J. Path. 144:337-347, 1994., Snow A D, et al. Arch. Biochem. Biophys. 320:84-95, 1995.), syndecan-2 (Lashley T, et al. Neuropath. Appl. 32:492-504, 2006., Verbeek M M, et al. Am. J. Path. 155:2115-2125,1999), agrin (Cotman S L, et al. Mol Cell. Neurosc. 15:183-198, 2000., Lashley T, et al. Neuropath. Appl. 32:492-504, 2006, Schultz J G, et al. Europ. J. Neuorsc. 10:2085-2093, 1998, Verbeek M M, et al. Am. J. Path. 155:2115-2125,1999, Watanabe N, et al. FASEB J. published online, Apr. 14, 2004., Watson D J, et al. J. Biol. Chem. 272:31617-31624, 1997.) and glypican (Lashley T, et al. Neuropath. Appl. 32:492-504, 2006., Schultz J G, et al. Europ. J. Neuorsc. 10:2085-2093, 1998, Verbeek M M, et al. Am. J. Path. 155:2115-2125,1999., Watanabe N, et al. FASEB J. published online, Apr. 14, 2004., Watson D J, et al. J. Biol. Chem. 272:31617-31624, 1997.) have been specifically immunolocalized to amyloid plaques in AD brain. In addition, our studies have identified these same HSPGs in the amyloid plaque deposits in APP mouse transgenic brain (FIG. 1) (Cummings J A, et al. Annual Meeting of Neuroscience, Washington, DC, November 2005, 102, Snow A D, et al. 8th International Conference on Alzheimer's and Parkinson's disease, Salzburg, Austria, March 2007). Sulfated GAGs and polyanions also play a role in PHF formation such as observed in NFTs in AD brain. In early studies by Snow et al (Snow A D, et al. Acta Neuropath. 78:113-123, 1989.) cationic dyes retained PGs in tissues and at the electron microscopic level it was clear that PGs were specifically co-localized to the PHFs in NFTs, in a specific periodic fashion. HSPG antibodies also immunolocalized HSPGs to tangles in AD brain (Goedert M, et al. Nature 383:550-553, 1996, Snow A D, and T N Wight. Neurobiol. Aging 10:481-497, 1989., Snow A D, et al. Am. J. Path. 137:1253-1270, 1990., Snow A D and G M Castillo. Amyloid: Int. J. Exp. Clin. Invest. 4:135-141, 1997.). Evidence by a number of groups later confirmed that highly sulfated GAGs (i.e. heparan sulfate and heparin) were potent inducers of tau polymerization into PHFs (Friedhoff P, et al., Biochem. 37:10223-10230, 1998. Goedert M, et al. Nature 383:550-553, 1996, Hasegawa M, et al. J. Biol. Chem. 272:33118-33124, 1997, Perez M, et al. J. Neurochem. 67:1183-1190, 1996). Since heparin is only found primarily in mast cells (not in brain tissue), it is postulated that the heparan sulfate class of PGs are important in the induction of PHFs as observed in AD brain.
Syndecan-2 is widely expressed in many tissues including brain. In neurons, syndecan-2 is concentrated at synapses in dimer/multimer clusters playing an essential role in creating specialized membrane environments for post-synaptic signaling (Ethell I M, et al., Neuron 31:1001-1013, 2001). The human syndecan-2 transcript consists of 5 exons, coding for a 22 kDa protein product that has 201 residues. The first of the GAG attachment sites in syndecan-2 is encoded by exon 2 and the other 2 GAG attachment sites, representing adjacent duplicate SG amino acid residues with a flanking cluster of acidic residues encoded by the combined sequence derived from the boundary of exons 2/3.
Agrin is also a large PG with the gene encoding a protein with a predicted MW of 225 kDa. At least 3 HS GAG attachment sites are present in the amino-terminal half of agrin (Hoch W, et al., EMBO J. 13:2814-2821, 1994., Tsen G, et al., J. Biol. Chem. 270:3392-3399, 1995.). The extensive glycosylation in this region increases the apparent molecular mass of agrin to 600 kDa. The C-terminal half of agrin is active in acetylcholine receptor aggregation and contains binding sites for dystroglycan, heparin and some integrins (Bezakova G, and M A Ruegg, Nat. Rev. Mol. Cell Biol. 4:295-308, 2003.). Agrin is expressed as several isoforms in various tissues.
Six different glypicans have been identified in mammals (Esko J D, and S B Selleck, Ann. Rev. Biochem. 71:435-471, 2002.); they are encoded by 6 independent genes that contain 8-12 exons. All glypicans are approximately 60-70 kDa in size. The GAG attachment sites are usually identified as a cluster, which locate within the last 50 residues at the C-terminus, next to a glyosylphosphotidy-linositol membrane anchor (Kramer K L, and H J Yost , Ann. Rev. Gen. 37:461-484, 2003., Veugelers M, et al., J. Biol. Chem., 274:26969-26977, 1999.).
It is believed that HSPGs facilitate Aβ to ultimately adapt a beta-sheet conformation and into insoluble amyloid fibrils. Consistent with this hypothesis, HSPGs such as perlecan (Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883, 1991., Snow A D, et al. J. Histochem. Cytochem. 40:105-113, 1992. , Snow A D, et al. Arch. Biochem. Biophys. 320:84-95, 1995.), agrin (Dolan M, et al. J. Biol. Chem. 272:4316-4322, 1997.) and glypican (Watson D J, et al. J. Biol. Chem. 272:31617-31624, 1997.) can bind with high affinity to AB and APPs (Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883, 1991). In addition, HSPGs, such as perlecan, enhance fibrillar Aβ amyloid deposition and persistence in brain, when co-infused with Aβ into rodent hippocampus (Snow A D, et al. Neuron 12: 219-234, 1994.). Furthermore, perlecan and HS GAGs can induce Aβ 1-40 peptides in vitro to adopt a congophilic Maltese-cross spherical plaque core appearance identical to that observed in AD brain (Choi P Y, et al. Neuroscience Meeting, Orlando, Fla., November 2002., Snow A D, et al. 10th International Symposium on Amyloid and Amyloidosis, Tours, France, April 2004.). These studies implicate HSPGs as important co-factors that may lead to the accumulation and persistence of Aβ. Studies indicate that the highly sulfated GAG chains (and not the core protein) are critical for formation and acceleration of Aβ amyloid (as observed in “plaques”) (Castillo G M, et al. J. Neurochem. 72:1681-1687, 1999), and for tau protein to form PHFs (as observed in “tangles”) (Friedrich M V, et al. J. Biol. Chem. 294:259-270, 1999, Goedert M, et al. Nature 383:550-553, 1996, Hasegawa M, et al. J. Biol. Chem. 272:33118-33124, 1997, Perez M, et al. J. Neurochem. 67:1183-1190, 1996). In one study, heparin/HS GAGs in which the sulfate moieties had been removed, demonstrated a nearly complete loss of the GAG's ability to accelerate Aβ amyloid fibril formation (Castillo G M, et al. J. Neurochem. 72:1681-1687, 1999). Thus it is postulated that any increase in HS GAG number, leads to an overall increase in GAG sulfation, which is critical to cause a formation and acceleration of both Aβ amyloid fibril and PHF formation in AD. Studies are therefore needed that characterize the degree of sulfation in PG GAGs and elucidate the role of sulfation in Aβ amyloid fibril and PHF formation in AD.
Amyloid as a Therapeutic Target for Alzheimer's Disease
Alzheimer's disease is characterized by the deposition and accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein, Aβ or β/A4 (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890, 1984; Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249, 1985; Husby et al., Bull. WHO 71:105-108, 1993). Aβ is derived by protease cleavage from larger precursor proteins termed β-amyloid precursor proteins (APPs) of which there are several alternatively spliced variants. The most abundant forms of the APPs include proteins consisting of 695, 751 and 770 amino acids (Tanzi et al., Nature 31:528-530, 1988).
The small Aβ peptide is a major component that makes up the amyloid deposits of “plaques” in the brains of patients with Alzheimer's disease. In addition, Alzheimer's disease is characterized by the presence of numerous neurofibrillary “tangles”, consisting of paired helical filaments which abnormally accumulate in the neuronal cytoplasm (Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986; Kosik et al., Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986; Lee et al., Science 251:675-678, 1991). The pathological hallmark of Alzheimer's disease is therefore the presence of “plaques” and “tangles”, with amyloid being deposited in the central core of the plaques. The other major type of lesion found in the Alzheimer's disease brain is the accumulation of amyloid in the walls of blood vessels, both within the brain parenchyma and in the walls of meningeal vessels that lie outside the brain. The amyloid deposits localized to the walls of blood vessels are referred to as cerebrovascular amyloid or congophilic angiopathy (Mandybur, J. Neuropath. Exp. Neurol. 45:79-90, 1986; Pardridge et al., J. Neurochem. 49:1394-1401, 1987)
For many years there has been an ongoing scientific debate as to the importance of “amyloid” in Alzheimer's disease, and whether the “plaques” and “tangles” characteristic of this disease were a cause or merely a consequence of the disease. Within the last few years, studies now indicate that amyloid is indeed a causative factor for Alzheimer's disease and should not be regarded as merely an innocent bystander. The Alzheimer's Aβ protein in cell culture has been shown to cause degeneration of nerve cells within short periods of time (Pike et al., Br. Res. 563:311-314, 1991; J. Neurochem. 64:253-265, 1995). Studies suggest that it is the fibrillar structure (consisting of a predominant β-pleated sheet secondary structure), characteristic of all amyloids, that is responsible for the neurotoxic effects. Aβ has also been found to be neurotoxic in slice cultures of hippocampus (Harrigan et al., Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death in transgenic mice (Games et al., Nature 373:523-527, 1995; Hsiao et al., Science 274:99-102, 1996). Injection of the Alzheimer's Aβ into rat brain also causes memory impairment and neuronal dysfunction (Flood et al., Proc. Natl. Acad. Sci. USA 88:3363-3366, 1991; Br. Res. 663:271-276, 1994).
Probably, the most convincing evidence that Aβ amyloid is directly involved in the pathogenesis of Alzheimer's disease comes from genetic studies. It was discovered that the production of Aβ can result from mutations in the gene encoding, its precursor, β-amyloid precursor protein (Van Broeckhoven et al., Science 248:1120-1122, 1990; Murrell et al., Science 254:97-99, 1991; Haass et al., Nature Med. 1:1291-1296, 1995). The identification of mutations in the beta-amyloid precursor protein gene that cause early onset familial Alzheimer's disease is the strongest argument that amyloid is central to the pathogenetic process underlying this disease. Four reported disease-causing mutations have been discovered which demonstrate the importance of Aβ in causing familial Alzheimer's disease (reviewed in Hardy, Nature Genet. 1:233-234, 1992). All of these studies suggest that providing a drug to reduce, eliminate or prevent fibrillar Aβ formation, deposition, accumulation and/or persistence in the brains of human patients will serve as an effective therapeutic.
Modulators of APP Secretases as Therapeutic Targets for Alzheimer's Disease
Elucidating APP metabolism and its role in the formation of Aβ plaques in AD is becoming increasingly important as therapeutics for AD and other beta-amyloid protein diseases are sought. Intracellular trafficking and proteolytic processing of APP directly influences the amount and type of Aβ peptide and can thus have a profound impact on amyloid plaque load.
Processing of APP in vivo and in cultured cells occurs by two major pathways (Haass and De Strooper, Science 286(5441):916-9 (1999) and; Selkoe, Physiol Rev. 81(2):741-66, (2001)). Cleavage of APP at the N-terminus of the Aβ region by β-secretase and at the C-terminus by γ-secretases represents the amyloidogenic pathway for processing of APP. β-secretase cleaves APP between residues Met595 and Asp596 (codon numbering refers to the APP695 isoform), and yields Aβ peptide plus the β-C-terminal fragment (βCTF or C99). Following β-secretase cleavage, a second cleavage by γ-secretase occurs at the C-terminus of Aβ peptide that releases Aβ from CTF. This cleavage occurs in the vicinity of residue 636 of the C-terminus. γ-secretase can cleave the C-terminal region at either Val636 or Ile638 to produce a shorter Aβ peptide (Aβ1-40) or the longer Aβ peptide (Aβ1-42). The predominant form of Aβ found in the cerebrospinal fluid and conditioned media of cultured cells is the shorter Aβ40 peptide. Despite its lower abundance, Aβ42 is the peptide that is initially deposited within the extracellular plaques of AD patients. In addition, Aβ42 is shown to aggregate at a much lower concentration than the Aβ40 form. APP can alternatively be processed via a non-amyloidogenic pathway where α-secretase cleaves within the Aβ domain between Lys611 and Leu612, and produces a large soluble α-APP domain (sAPPα) and a α-C-terminal fragment (αCTF or C83). The latter can then be cleaved by γ-secretase at residue 636 or 638 to release a P3 peptide and the APP intracellular domain (AICD). The α-cleavage pathway is the major pathway used to process APP in vivo; it does not yield Aβ peptide (Selkoe, Physiol Rev. 81(2):741-66, (2001). The characterization of APP cleavage and the related secretases has provided significant advancement in therapeutic strategies that may lead to limiting the deposition of Aβ peptide in the brain, and eliminate or delay the associated pathological effects in AD.